Neuroblastoma (NB) is an extra-cranial pediatric cancer. Amplification of the proto-oncogene MYCN is associated with advanced stage, high risk NB and poor prognosis. Novel drugs and alternative treatments are being investigated for patients with advanced NB with MYCN amplification. However, finding an effective treatment strategy for advanced stage NB remains elusive. In order to address this limitation, the proposed project examines the effects of calcium and magnesium signaling on cell migration and invasion in NB cells with different MYCN status. The preliminary data indicate that MYCN initiates a change in calcium signaling, accompanied by a change in the transcriptional regulation of calcium channels, namely Orai1, Orai3 and TRPM7. In addition, MYCN induces calcium-dependent NB cell migration. These observations support the hypothesis that MYCN alters calcium and magnesium homeostasis during cell migration and invasion through a mechanism that involves calcium-mediated regulation of integrin signaling and focal adhesion turnover, and calcium- and magnesium-permeable ion channels. The research plan has the following specific aims: (1) Examine the effect of MYCN on [Ca2+] and [Mg2+] homeostasis in NB cells. NB cells with different MYCN status will be cultured in external media with different extracellular [Ca2+]/[Mg2+] ratios. The intracellular calcium and magnesium will be measured, and migration and invasion rates will be determined in these cells. (2) Elucidate the mechanism by which MYCN regulation of calcium and magnesium promotes NB migration and invasion. The effect of calcium and magnesium on the expression and phosphorylation of focal adhesion kinase and integrin, focal adhesion turnover rate (assembly, disassembly and recycling of focal adhesion proteins), and actin cytoskeletal re-organization will be examined in NB cells that are actively migrating. (3) Identify the molecular components regulating NB migration and invasion. The calcium- permeable ORAI channels and the calcium- and magnesium-permeable TRPM7 channel protein will be modulated pharmacologically or using molecular methods (RNAi or transient over-expression), and the effect of these changes on cell migration and invasion, focal adhesion turnover (assembly, disassembly, and recycling of focal adhesion proteins), and intracellular [Ca2+] will be determined in NB cells. This study may identify new biomarkers for advanced stage NB, and reveal novel targets for the development of more effective chemotherapeutic drugs. In addition, this study may lead to a new model of the regulation of NB progression, which includes monitoring the status of the prognostic marker MYCN as well as the dietary intake, metabolism and cellular regulation of calcium and magnesium. This new model may apply to other cancers with MYC amplification (e.g. breast and prostate cancers, and lymphoma, etc).